The yeast diadenosine and diphosphoinositol polyphosphate phosphohydrolase DDP1 is a Nudix enzyme with pyrophosphatase activity on diphosphoinositides, dinucleotides, and polyphosphates. These substrates bind to diverse protein targets and participate in signaling and metabolism, being essential for energy and phosphate homeostasis, ATPase pump regulation, or protein phosphorylation. An exhaustive structural study of DDP1 in complex with multiple ligands related to its three diverse substrate classes is reported. This allowed full characterization of the DDP1 active site depicting the molecular basis for endowing multisubstrate abilities to a Nudix enzyme, driven by phosphate anchoring following a defined path. This study, combined with multiple enzyme variants, reveals the different substrate binding modes, preferences, and selection. Our findings expand current knowledge on this important structural superfamily with implications extending beyond inositide research. This work represents a valuable tool for inhibitor/substrate design for ScDDP1 and orthologs as potential targets to address fungal infections and other health concerns.

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Inositol pyrophosphates are important biomolecules associated with apoptosis, cell growth and kinase regulation, yet their exact biological roles are still emerging and probes do not exist for their selective detection....

Chiral sugar derivatives are potential cyclitol surrogates of the Ca 2+ -mobilizing intracellular messenger D-myoinositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ]. Six novel polyphosphorylated analogues derived from both D-and L-glucose were synthesized. Binding to Ins(1,4,5)P 3 receptors [Ins(1,4,5)P 3 R] and the ability to release Ca 2+ from intracellular stores via type 1 Ins(1,4,5)P 3 Rs were investigated. β-D-Glucopyranosyl 1,3,4-trisphosphate, with similar phosphate regiochemistry and stereochemistry to Ins(1,4,5)P 3 , and α-D-glucopyranosyl 1,3,4-trisphosphate are full agonists, being equipotent and 23-fold less potent than Ins(1,4,5)P 3 , respectively, in Ca 2+ -release assays and similar to Ins(1,4,5)P 3 and 15-fold weaker in binding assays. They can be viewed as truncated analogues of adenophostin A and refine understanding of structure-activity relationships for this Ins(1,4,5)P 3 R agonist. L-Glucose-derived ligands, methyl α-Lglucopyranoside 2,3,6-trisphosphate and methyl α-L-glucopyranoside 2,4,6-trisphosphate, are also active, while their corresponding D-enantiomers, methyl α-D-glucopyranoside 2,3,6-trisphosphate and methyl α-D-glucopyranoside 2,4,6-trisphosphate, are inactive. Interestingly, both L-glucose-derived ligands are partial agonists: they are among the least efficacious agonists of Ins(1,4,5)P 3 R yet identified, providing new leads for antagonist development.